Ochratoxin A Contamination of California Pistachios and Identification of Causal Agents.

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium spp., which contaminates many crops, including pistachios. Pistachios contaminated with OTA may be subjected to border rejections resulting in significant economic losses to the United States agricultural revenues. The current study examined prevalence of OTA in California-grown pistachios and identified its causal agents. OTA was detected in 20% of samples from 2018 to 2021 (n = 809), with 18% of samples exceeding the European Union regulatory limit of 5 µg/kg. Fungi potentially responsible for OTA contamination were isolated from leaves, nuts, and soil collected from 14 pistachio orchards across California. A total of 1,882 isolates of Aspergillus section Nigri and 85 isolates of section Circumdati were recovered. Within section Nigri, 216 (11.5%) isolates were identified as potential OTA producers using a boscalid-resistance assay. Phylogenetic analyses of partial gene sequences for ß-tubulin and calmodulin genes resolved section Circumdati into four species: A. ochraceus (33%), A. melleus (28%), A. bridgeri (21%), and A. westerdijkiae (19%). A. westerdijkiae produced the highest levels of OTA in inoculated pistachios (47 µg/g), followed by A. ochraceus (9.6 µg/g) and A. melleus (3.3 µg/g). A. bridgeri did not produce OTA. OTA production by section Circumdati was optimal from 20 to 30°C. All 216 boscalid-resistant isolates from section Nigri were identified as A. tubingensis, and representative isolates (n = 130) produced 3.8 µg/kg OTA in inoculated pistachios. This is the first detailed report on OTA contamination and causal fungi in California pistachios and will be helpful in devising effective management strategies.

Genomic regions associated with virulence in Setosphaeria turcica identified by linkage mapping in a biparental population.

Northern corn leaf blight (NCLB) and sorghum leaf blight (SLB) are significant diseases of maize and sorghum, respectively, caused by the filamentous fungus Setosphaeria turcica. Strains of S. turcica are typically host-specific and infect either maize or sorghum. Host specificity in this pathogen is attributed to a single locus for maize and a second distinct locus for sorghum. To identify the genetic basis of host specificity in S. turcica, we generated a biparental population of S. turcica by crossing strains specific to maize and sorghum, phenotyped the population for leaf blight on sorghum and maize, genotyped the population to create a linkage map of S. turcica, and located candidate virulence regions. A total of 190 ascospores from 35 pseudothecia were isolated from the cross of maize and sorghum-specific strains. Greenhouse phenotyping of the biparental population (n = 144) showed independent inheritance of virulence, as indicated by a 1:1:1:1 segregation for virulence to maize, sorghum, both maize and sorghum, and avirulence to both crops. The population and host-specific parent strains were genotyped using genome skim sequencing on an Illumina NovaSeq 6000 platform resulting in over 780 million reads. A total of 32,635 variants including single nucleotide polymorphisms and indels were scored. There was evidence for a large deletion in the sorghum-specific strain of S. turcica. A genetic map consisting of 17 linkage groups spanning 3,069 centimorgans was constructed. Virulence to sorghum and maize mapped on distinct linkage groups with a significant QTL detected for virulence to maize. Furthermore, a single locus each for the in vitro traits hyphal growth rate and conidiation were identified and mapped onto two other linkage groups. In vitro traits did not correlate with in planta virulence complexity, suggesting that virulence on both hosts does not incur a fitness cost. Hyphal growth rate and conidiation were negatively correlated, indicating differences in hyphal growth versus dispersal ability for this pathogen. Identification of genetic regions underlying virulence specificity and saprotrophic growth traits in S. turcica provides a better understanding of the S. turcica- Andropogoneae pathosystem.

Genetic Diversity of Aspergillus flavus Associated with Chili in Nigeria and Identification of Haplotypes with Potential in Aflatoxin Mitigation.

Dried red chili (Capsicum spp.), a widely produced and consumed spice in Nigeria, is often contaminated by aflatoxins. Aflatoxins are potent mycotoxins of severe health and economic concern worldwide. Aspergillus flavus often contaminates crops with aflatoxins in warm regions; however, not all isolates are aflatoxin producers. Nonaflatoxigenic isolates have potential as biocontrol agents for aflatoxin mitigation. The current study examined the genetic diversity of A. flavus (n = 325) associated with chilies in Nigeria and identified 123 nonaflatoxigenic isolates. The Nigerian A. flavus isolates from chili were diverse at 17 microsatellite loci, with 5 to 36 alleles per locus, and included 152 haplotypes. The isolates that are active ingredients in Aflasafe, registered for aflatoxin biocontrol on maize and groundnuts in Nigeria, did not share haplotypes with the chili isolates. Of the 152 haplotypes, 65% produced aflatoxins in autoclaved maize, some of which (17%) produced >100,000 µg/kg of aflatoxins. Aflatoxins were not detected in 35% of the haplotypes. Cluster amplification pattern assay detected large deletions in the aflatoxin biosynthetic clusters of some (32%) of the nonaflatoxigenic haplotypes. Coinfection of chili with nonaflatoxigenic isolates from chilies (n = 7) and A. aflatoxiformans resulted in a significantly greater average reduction in total aflatoxins compared with that achieved by Aflasafe active ingredient isolates (P < 0.01). These nonaflatoxigenic isolates are a genetic resource for the development of biological control products for aflatoxin mitigation in chilies in Nigeria and should be evaluated under field conditions.

Phenotypic Differentiation of Two Morphologically Similar Aflatoxin-Producing Fungi from West Africa.

Aflatoxins (AF) are hepatocarcinogenic metabolites produced by several Aspergillus species. Crop infection by these species results in aflatoxin contamination of cereals, nuts, and spices. Etiology of aflatoxin contamination is complicated by mixed infections of multiple species with similar morphology and aflatoxin profiles. The current study investigates variation in aflatoxin production between two morphologically similar species that co-exist in West Africa, A. aflatoxiformans and A. minisclerotigenes. Consistent distinctions in aflatoxin production during liquid fermentation were discovered between these species. The two species produced similar concentrations of AFB1 in defined media with either urea or ammonium as the sole nitrogen source. However, production of both AFB1 and AFG1 were inhibited (p < 0.001) for A. aflatoxiformans in a yeast extract medium with sucrose. Although production of AFG1 by both species was similar in urea, A. minisclerotigenes produced greater concentrations of AFG1 in ammonium (p = 0.039). Based on these differences, a reliable and convenient assay for differentiating the two species was designed. This assay will be useful for identifying specific etiologic agents of aflatoxin contamination episodes in West Africa and other regions where the two species are sympatric, especially when phylogenetic analyses based on multiple gene segments are not practical.

Molecular Analysis of S-morphology Aflatoxin Producers From the United States Reveals Previously Unknown Diversity and Two New Taxa.

Aflatoxins are highly toxic carcinogens that detrimentally influence profitability of agriculture and the health of humans and domestic animals. Several phylogenetically distinct fungi within Aspergillus section Flavi have S-morphology (average sclerotial size < 400 µm), and consistently produce high concentrations of aflatoxins in crops. S-morphology fungi have been implicated as important etiologic agents of aflatoxin contamination in the United States (US), but little is known about the diversity of these fungi. The current study characterized S-morphology fungi (n = 494) collected between 2002 and 2017, from soil and maize samples, in US regions where aflatoxin contamination is a perennial problem. Phylogenetic analyses based on sequences of the calmodulin (1.9 kb) and nitrate reductase (2.1 kb) genes resolved S-morphology isolates from the US into four distinct clades: (1) Aspergillus flavus S-morphotype (89.7%); (2) Aspergillus agricola sp. nov. (2.4%); (3) Aspergillus texensis (2.2%); and (4) Aspergillus toxicus sp. nov. (5.7%). All four S-morphology species produced high concentrations of aflatoxins in maize at 25, 30, and 35°C, but only the A. flavus S-morphotype produced unacceptable aflatoxin concentrations at 40°C. Genetic typing of A. flavus S isolates using 17 simple sequence repeat markers revealed high genetic diversity, with 202 haplotypes from 443 isolates. Knowledge of the occurrence of distinct species and haplotypes of S-morphology fungi that are highly aflatoxigenic under a range of environmental conditions may provide insights into the etiology, epidemiology, and management of aflatoxin contamination in North America.

Characterization of Aspergilli from dried red chilies (Capsicum spp.): Insights into the etiology of aflatoxin contamination.

Aflatoxins are toxic carcinogens produced by several species of Aspergillus section Flavi, with some aflatoxin producers associated with specific crops. Red chilies (Capsicum spp.) are grown in warm regions that also favor aflatoxin-producers. Aflatoxins in red chilies may result in serious health concerns and severe economic losses. The current study sought to gain insight on causal agents of aflatoxin contamination in red chilies. Naturally contaminated chilies from markets in Nigeria (n = 55) and the United States (US) (n = 169) were examined. The A. flavus L strain was the predominant member of Aspergillus section Flavi (84%) in chilies. Highly toxigenic fungi with S strain morphology were also detected in chilies from both countries (11%), followed by A. tamarii (4.6%) and A. parasiticus (0.4%). Fungi with L morphology produced significantly lower quantities of aflatoxins (mean = 43 µg g-1) compared to S morphology fungi (mean = 667 µg g-1; p < 0.01) in liquid fermentation. Eighty-one percent of S morphology fungi from chilies in US markets produced only B aflatoxins, whereas 20%, all imported from Nigeria, produced both B and G aflatoxins; all S morphology fungi from Nigerian chilies produced both B and G aflatoxins. Multi-gene phylogenetic analyses of partial gene sequences for nitrate reductase (niaD, 2.1 kb) and the aflatoxin pathway transcription factor (aflR, 1.9 kb) resolved Aspergilli recovered from chilies into five highly supported distinct clades: 1) A. parasiticus; 2) A. flavus with either L or S morphology; 3) A. minisclerotigenes; 4) A. aflatoxiformans, and 5) a new lineage. Aspergillus aflatoxiformans and the new lineage produced the highest concentrations of total aflatoxins in chilies, whereas A. flavus L strains produced the least. The results suggest etiology of aflatoxin contamination of chili is complex and may vary with region. Knowledge of causal agents of aflatoxin contamination of chilies will be helpful in developing mitigation strategies to prevent human exposure.

Aspergillus texensis: A Novel Aflatoxin Producer with S Morphology from the United States.

Aflatoxins are carcinogenic metabolites produced primarily by fungi within Aspergillus section Flavi. These fungi infect a wide range of crops in warm regions. Molecular phylogenetic analyses of fungi with S morphology (average sclerotium size < 400 µm) within section Flavi collected from across the United States (US) resulted in the discovery of a novel aflatoxin-producing species, Aspergillus texensis. Aspergillus texensis was isolated from maize grown in Arkansas, Louisiana, and Texas, and from soils cropped to maize in Texas. Aspergillus texensis produces sparse conidia and abundant sclerotia on various culture media, and on maize. Physiological studies have revealed optimal growth on culture media at 35 °C. All isolates of A. texensis produced B and G aflatoxins, cyclopiazonic acid and aspergillic acid. Aspergillus texensis and A. flavus S strain morphotypes produced similar concentrations of total aflatoxins on maize (p > 0.05). Phylogenetic analyses of aflatoxin-producers based on partial gene sequences of the ß-tubulin (0.9 kb), calmodulin (1.2 kb), and nitrate reductase (2.1 kb) genes placed A. texensis in a highly supported monophyletic clade closely related to A. minisclerotigenes and a previously reported unnamed lineage designated Lethal Aflatoxicosis Fungus.